Summary of survey results showing 16 high-burden TB (HBCs) and/or high-burden multi-drug resistant TB countries' (HBC-MDRs) perspectives on retooling national TB programmes with seven new TB diagnostic tools.

<p><b>Legend.</b> TB: tuberculosis. NTP: national TB programme. NRL: national TB reference laboratory. QA: quality assurance. Results were obtained through questionnaires (and interviews where indicated).</p

Order in which different laboratory tests for identification of bacteria of the <i>Mycobacterium tuberculosis</i> complex in positive culture isolates are performed in national TB reference laboratories of 15 high endemic countries.

<p>NRL: national TB reference laboratory, AFB: acid fast bacilli, MTB: <i>Mycobacterium tuberculosis</i>.</p>*<p>NRL indicated the type of tests performed, but not the order in which these tests were performed.</p

Procurement and logistics of immunochromatographic tests for identification of bacteria of the <i>Mycobacterium tuberculosis</i> complex in positive culture isolates in national TB reference laboratories of 15 high endemic countries.

<p>NRL: national TB reference laboratory, ICT: immunochromatographic test, Y: Yes, N: No, DNK: Did not know, n.a.: not applicable, Room: Room temperature, Tauns: Capilia TB assay (Tauns, Japan), SD: TB Ag MPT64 Rapid assay (SD Bio Standard Diagnostic, India/South Korea), BD: BD MGITTM TBc Identification Test (BD Diagnostics, USA).</p>1<p>List of countries eligible for negotiated pricing schemes for immunochromatic speciation tests as shown on the Foundation for Innovative New Diagnostics (FIND) website (<a href="http://www.finddiagnostics.org/about/what_we_do/successes/find-negotiated-prices/bactec-mgit.html" target="_blank">http://www.finddiagnostics.org/about/what_we_do/successes/find-negotiated-prices/bactec-mgit.html</a>).</p>2<p>Also mentioned Genotype MTBDRplus (Hain, Germany) and GeneXpert (Cepheid, USA).</p>3<p>Mentioned AccuProbe (GenProbe, USA).</p

Diagnostic development pipeline.

<p>The status of the pipeline for new diagnostics for tuberculosis in July 2011 and the placement of liquid culture and rapid speciation tests within it. Reproduced from ‘Global tuberculosis control: WHO report 2011’ with permission.</p

TB associated cytokines after 4 and 6 weeks of infection.

<p>IL-17 (<b>A</b>) and IL-6 (<b>B</b>) are significantly increased in the infected mice (open symbols) compared with uninfected mice (black symbols) at 4 weeks after infection. At 6 weeks post infection levels in all infected and non-infected mice were similar and around the detection limit for both cytokines. IFNγ (<b>C</b>), IP-10 (<b>D</b>)and MIG (<b>E</b>) are increased both at 4 and 6 weeks after infection compared to non-infected mice. As MIG levels were higher in all infected mice than in all uninfected mice except 1, at 6 weeks significance was not reached (p = 0.08). Levels of MCP-1 (<b>F</b>) were increased in infected mice at 4 weeks post infection compared to uninfected mice. At both 4 and 6 weeks post infection the range of MCP-1 levels was quite wide between both infected and uninfected mice and levels at 6 weeks were very similar between infected and uninfected mice. * p<0.05, ** p<0.01, *** p<0.001</p

Schematic representation of the expected release of TB antigens and consequential host antibody response upon initiation of treatment.

<p>We propose that measurement of these increased levels can be useful for diagnosing TB in a treat-to-test strategy.</p

Changes in cytokine levels during TB treatment.

<p>IFNγ, IP-10, MIG and MCP-1 levels were measured in infected (left hand panels, A, C, E, G) and non-infected (right hand panels, B, D, F, H) mice before (black squares) and after 3, 7, and 21 days of treatment with RIF+INH (open circles) or placebo (black circles). In the infected groups within 7–21 days of treatment with RIF+INH levels of all 4 cytokines were lower compared to placebo treated mice. In the non-infected mice no differences in levels were seen between different treatments or time points. * p<0.05, ** p<0.01, *** p<0.001</p

Lung CFU and relative lung weights of mice.

<p><b>A</b>: Relative lung weights were calculated as a percentage of total body mass (mean +− SE). Black squares: uninfected placebo, open squares: uninfected RIF+INH treated, black circles: infected placebo, open circles: infected RIF+INH treated mice. At start of treatment, relative lung weights were increased in infected mice compared to uninfected mice (p<0.001). After an initial increase within 3 days, relative lung weight started to decrease within 3–7 days after start of treatment with RIF+INH in the infected mice although remaining higher than in the uninfected mice until the end of the experiment (p<0.01). Treatment had no effect on lung weight in uninfected mice. <b>B</b>: CFU counts of lung homogenates of sacrificed mice. Median baseline level in infected mice prior to treatment was 1.3×10⁶CFU per lung (black squares) and similar levels were found after 7 and 21 days of placebo treatment (black circles, 3 days not available due to contamination of culture plates). Treatment with RIF+INH strongly reduced CFU counts within 7 days (open circles; detection limit 2×10³ CFU/lung). Counts from 1 mouse are not available due to contamination. Cultures of undiluted lung homogenates from uninfected mice yielded no TB colonies (not shown). ** p<0.01</p

Results of hybridiations of, 31 Middlebrook cultures, 13 MGIT cultures, and 4 controls, to the 9 bead array.

<p>Fluorescence intensity in arbitrary units is indicated on the left hand side as determined by the MAGPIX machine, this data is visualized in the form of a “line probe assay” (Excel 2010, Microsoft, Seattle, USA) on the right hand side where the % of the total signal in each assay resulting from each specific bead is indicated as a grey scale (where <10% of total signal from a bead species is white, and >30% of total signal from a bead species is black).</p

Effect of increasing lysis time on the yield of rRNA from an NTM (<i>M. avium</i>) and an <i>M. tuberculosis</i> culture.

<p>Solid lines and filled triangles/diamonds indicate the species specific bead signal, dashed lines and filled squares represent the 23S rRNA control signal, empty triangles indicate the average signal from the beads targeting other species (error bars +− one standard deviation). Lysis was performed at 30 Hz in the presence of Zirconium beads for pulses of 10 minutes. Aliquots of the prepared mycobacteria were removed after each period of shaking collected and analysed together in the rRNA capture MAGPIX assay. Upper graph <i>M. avium</i> lower graph <i>M. tuberculosis</i>.</p